This invention relates generally to fluorescein derivatives, and more particularly, relates to 5(6)-methyl substituted fluorescein derivatives and their production by the reduction of 5(6)-carboxyfluorescein to 5(6)-methyl substituted fluorescein.
There are numerous fluorescein derivatives that have functional groups which are suitable for reaction with other molecules. Many of these derivatives have been used commercially in the production of tracers for analytical applications which range from the probing of cell functions to the monitoring of the level of one or more drugs in physiological test samples. See, for example, C. Dive et al., Molecular and Cellular Probes 2:31 (1988); M. L. Graber et al., Anal. Biochem. 156:202 (1986); P. J. Brynes et al., U.S. Pat. No. 4,869,132; and N. Y. Wang et al., European Patent Application Publ. No. EP264797 (1988). Examples of such applications include the Fluorescence Polarization Immunoassay (FPIA) for use on commercially available instruments, such as the Abbott AD.sub.x .RTM. instrument and the Abbott TD.sub.x .RTM. instrument (available from Abbott Laboratories, Abbott Park, Ill.). These derivatives include 5- and 6-carboxyfluorescein, 5- and 6-aminofluorescein and 4'-aminomethylfluorescein (M. T. Shipchandler, et. al., Anal. Biochem., 162:89 (1987). Of these, the 5- and 6-aminofluorescein compounds are the most difficult with which to work, since the amino group is not very nucleophilic. This difficulty is an expected consequence of the amino group being directly bound to the deactivating aromatic ring. The compound 4'-aminomethylfluorescein (4'-AMF) was an earlier attempt to solve this problem by adding a methylene group between the amino group and the aromatic ring. This addition succeeded in restoring normal amino group reactivity of 4'-AMF. However some 4'-AMF derivatives have been found to be unstable under the conditions of long term storage in aqueous buffers. Possibly this instability is due to a retro-Mannich reaction or subsequent elimination of the amino group (H. O. House, Modern Synthetic Reactions, 2nd Ed., 1972, pp 654-660). Additionally, the preparation and purification of 4'-AMF is tedious.
Martin et. al., (Cytometry 12:184-187 [1991]) report a preparation of 5-chloromethyl fluorescein diacetate by the halogenation of 5-methylfluorescein diacetate without including any experimental details. Also, Peerce et. al. (J. Biol. Chem. 260 (10):6026-31 [1985]) report a methyl substituted fluorescein derivative with the given structure: ##STR1## However, the experimental procedure reported by Peerce et al. would indicate that the compound was actually of the following structure: ##STR2## Khana et. al. (U.S. Pat. No. 4,439,365) disclose xanthene compounds of the following structures, but again offer no experimental details: ##STR3##
The reduction of 5(6)-carboxyfluorescein derivatives is not known. However, in general, carboxylic acid derivatives (i.e., free acids, metal salts, amides, esters, acid halides, etc.) may be reduced by a variety of methods. These methods have been reviewed extensively in the literature. See, for example, H. C. Brown, Boranes in Organic Chemistry, Cornell University Press (1972); and C. F. Lane, "Reductions of Organic Compounds with Diborane," Chemical Reviews 76:773-799 (1976). Also, the reduction of activated carboxylic acids to alcohols with sodium borohydride has been described. K. Ramasamy et al., Synthesis: 42 (1982).
The present invention solves the problem of amino reactivity at the 5 or 6 position by adding a methylene group between it and the aromatic ring. Additionally, the novel processes employed results in the availability of novel and useful derivatives available for use in tracer synthesis, and for use in analytical assays.